臺灣博碩士論文加值系統

腫瘤細胞黏著至血管內皮細胞以及隨後從血管穿越內皮細胞層轉移 (transendothelial migration) 到不同的組織/器官的過程，在腫瘤轉移中扮演一個重要的角色。腫瘤相關巨噬細胞就目前文獻上所知，可促進腫瘤的轉移與生長。本論文研究證明，巨噬細胞條件培養液 ( Macrophage conditioned medium, MCM) 可引起人類乳腺癌細胞株 (MCF-7) 和鼻咽癌細胞株 (NPC -TW01) 黏著至人類臍靜脈內皮細胞 (HUVECs) 的能力 (Cell-cell adhesion) 增加以及可促進穿越內皮細胞層轉移 (transendothelial migration) 的能力。MCM 可刺激 HUVECs、MCF-7及NPC-TW01 細胞產生更多的內皮素-1 (Endothelin-1, ET-1)(ET-1濃度：HUVECs＞MCF-7＞NPC-TW01)。另外，MCM 亦可增加 HUVECs 內皮素接受器B (ETR-B) 的表現以及增加 MCF-7和 NPC-TW01細胞內皮素接受器A (ETR-A)、內皮素接受器B (ETR-B)的表現。若分別添加內皮素接受器A拮抗劑 (BQ123)、內皮素接受器B拮抗劑 ( BQ788)以及ET-1 抗體則會阻斷 MCF-7和 NPC-TW01 細胞趨化、黏著至內皮細胞以及穿越內皮細胞層轉移的作用。此結果證明內皮素/內皮素接受器 (ET/ETRs) 之間的交互作用會影響 MCF-7、NPC-TW01 細胞和 HUVECs 間下游基因的訊號傳遞路徑。在我們的研究結果中亦發現，當我們加入了整合素 (Integrin) 的抗體時，會抑制 MCM 所引起的穿越內皮細胞層 (transendothelial migration) 的作用。若由 mRNA 實驗數據分析，ET/ETRs 會影響 MCF-7、NPC-TW01 細胞整合素aM,b1,b2,和 b3 的表現以及 HUVECs 整合素 aV, a5, b1, b2, 和 b3 和 ICAM-1, ICAM-2, VCAM-1, PE-CAM, E-selectin和P-selectin 的表現。此結果證明這些細胞黏著分子是乳腺癌細胞株、鼻咽癌細胞株進行內滲作用 (intravasation) 以及腫瘤轉移時所必須的。
加入介白素-8 接受器拮抗劑 (IL-8RA)，介白素-6可溶性接受器拮抗劑 (IL-6sR) 或腫瘤壞死因子可溶性的接受器拮抗劑 (sTNFR1) 以及分別加入對NFkB (nuclear factor-kappa B)，MEKK (MAP kinase )，p38MAPK，JNK (Jun N-terminal kinase) 的阻斷劑去分析經 MCM 培養過癌細胞的表現。由研究結果發現巨噬細胞引起癌細胞的 ETR-A/ETR-B表現是透過 IL-8、TNF-a、ERK (extracellularly regulated kinase) 和NFkB路徑。

Adhesion of tumor cells onto vascular endothelial cells and subsequent transendothelial migration are essential steps for cancer cells to enter in or exit from the vessels and thus metastasize to different tissues/organs. Tumor-associated macrophages have been known to provide many aids on tumor metastasis and malignant progression. In this study, we provide evidence demonstrating that macrophage conditioned medium (MCM) was able to induce human breast cancer MCF-7 and nasopharyngeal carcinoma NPC-TW01 cells to migrate toward and furthermore adhere onto endothelial cells, and subsequently induced cancer cells to achieve transendothelial migration. MCM induced endothelin (ET)-1 production in human umbilical vein endothelial cells (HUVECs) a then cancer cells. MCM also induced ET receptor (ETR)-B in endothelial cells and ETR-A and ETR-B in MCF-7 and NPC-TW01 cells. The ET antagonists (anti-ET-1 antibody, BQ123 and BQ788) could be used to block MCF-7 and NPC-TW01 cells chemotaxis toward endothelial cells, adhesion onto endothelial cells and transendothelial migration, suggesting that paracrine interactions between ETs and ETRs could induce signaling pathways and downstream gene expression to elicit more interactions between MCF-7 cells, NPC-TW01 cells and endothelial cells. Our data further indicated that ET axis induced expression of integrins aM,b1,b2, and b3 in MCF-7/NPC-TW01 cells and expression of integrins aV,a5,b1,b2, and b3 and the counter ligands of integrins, such as ICAM-1, ICAM-2, VCAM-1, PE-CAM, E-selectin, and P-selectin, in HUVECs. Because MCM-induced transendothelial migration of MCF-7 cells and NPC-TW01 cells could be drastically abolished by antagonizing antibodies against these integrins, the expression of these adhesion molecules was suggested to be required for tumor cells intravasation and furthermore cancer metastasis.
Anti-interleukin-8 receptor antagonizing antibody, interleukin-6 or tumor necrosis factor-a soluble receptor, and inhibitors against NF-B, MEKK, p38MAPK, and JNK, were used for analyzing the effects of these cytokines and signal pathways induced by MCM. Our data suggested that macrophages were able to elicit tumor necrosis factor-a-dependent ET-1 expression in HUVECs, and induced ETR-A/ETR-B expression in cancer cells via the interleukin-8,TNF-a,ERK,NF-kB pathway.

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目錄 I
圖表目錄 III
中文摘要 V
英文摘要 VII

第一章、緒論 1
一、 癌症侵入與轉移 (Tumor invasion and metastasis) 1
二、 腫瘤相關巨噬細胞 (Tumor associated macrophage , TAM) 2
三、 簡介乳癌 (Breast carcinoma) 3
四、 簡介鼻咽癌 (Nasopharyngeal carcinoma) 4
五、 簡介內皮素 (Endothelins) 4
六、 細胞黏著分子與癌症關係 7
七、 本實驗之目標 9
第二章、材料與方法 9
一、 實驗材料 11
1. 細胞株 11
2. 質體 11
3. 引子 11
4. 實驗藥品與試劑 11
二、 實驗儀器 16
三、 實驗方法 17
1. 人類乳癌細胞株培養 17
2. 人類鼻咽癌細胞株培養 18
3. 人類臍靜脈內皮細胞之繼代培養 18
4. 人類白血病細胞株培養以及製備巨噬細胞條件培養液 19
5. 反轉錄聚合酶連鎖反應 (RT-PCR) 20
6. 內皮素-1之酵素免疫測定法 (ELISA) 22
7. 流式細胞儀測定 ( Flow cytometric analysis) 23
8. 細胞趨化性分析 (Cell chemotaxis assay) 24
9. 細胞-細胞黏著分析 (Cell-to-cell adhesion assay) 24
10穿越內皮細胞層轉移分析 (Transendothelial migration
assay) 25
11. Luciferase reporter assay 活性測定 26
12. 西方墨點法(Western blot analysis) 28
13. 統計方法 31
第三章、實驗結果 32
一、 MCM 可促進 HUVECs 細胞中的 ET-1、ET-2、ETR-B 以及 MCF-7、NPC-TW01 細胞中的 ET-1、ET-2、ETR-A、ETR-B 之 mRNA 表現量增加 32
二、 MCM 可促進 HUVECs 細胞膜蛋白 ETR-B；MCF-7 細胞膜蛋白 ETR-A、ETR-B 以及 NPC-TW01 細胞膜蛋白 ETR-A 之表現量增加 33
三、 MCM 可促進 HUVECs、MCF-7 及 NPC-TW01 細胞中的 ET-1 的分泌 33
四、 以細胞趨化實驗 (Cell Chemotaxis Assay) 分析經 MCM 處理後的 MCF-7、NPC-TW01 細胞之細胞趨化有明顯增加的情形 34
五、 以 HUVECs 與腫瘤細胞黏著實驗 (Endothelial cell-tumor cell adhesion assay) 分析經 MCM 處理後的 MCF-7、NPC-TW01 細胞黏著於 HUVECs 的能力有明顯增加的情形 36
六、 以腫瘤細胞穿透內皮細胞層實驗 (Transendothelial migration assay) 分析經 MCM 處理後的 MCF-7、NPC-TW01細胞穿透 HUVECs 的能力有增加的情形 37
七、 MCM 可促進 MCF-7 及 NPC-TW01 中 Adhesion molecules 基因 之 mRNA 的表現量增加 38
八、 MCM 會增加 MCF-7 及 NPC-TW01 其細胞膜蛋白：Adhesion molecules 之表現量 39
九、 MCM 會增加 HUVECs 中 Adhesion molecules 基因 之 mRNA 的表現量 40
十、 以腫瘤細胞穿透內皮細胞層實驗 (Transendothelial migration assay) 分析 MCF-7、NPC-TW01 細胞經 MCM 前處理時，加入Adhesion molecules 抗體後，會抑制腫瘤細胞穿透 HUVECs 的能力 40
十一、 IL8-RA、IL6sR、sTNFR1 阻斷劑；PD98059、SP600125、SB202190和 NFkB 阻斷劑對 MCM 刺激 ET axis 表現的影響 41
十二、 以Luciferase 報告基因分析NFB 活性，以西方點墨法分析 ERK 及磷酸化 ERK 蛋白質表現 42
第四章、討論 44
第五章、實驗圖表 52
第六章、參考文獻 76
附錄 82

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